Methods and apparatus for sorting cells using an optical switch in a microfluidic channel network

ABSTRACT

Apparatus and Methods are provided for a microfabricated fluorescence activated cell sorter based on an optical switch for rapid, active control of cell routing through a microfluidic channel network. This sorter enables low-stress, highly efficient sorting of populations of small numbers of cells (i.e., 1000-100,000 cells). The invention includes packaging of the microfluidic channel network in a self-contained plastic cartridge that enables microfluidic channel network to macro-scale instrument interconnect, in a sterile, disposable format.

RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/928,650, filed Aug. 27, 2004, now U.S. Pat. No. 7,745,221, issuedJun. 29, 2010; which claims priority to U.S. Provisional Application No.60/499,294, filed Aug. 28, 2003, entitled “Microsorter Cartridge” andU.S. Provisional Application No. 60/574,897, filed May 26, 2004,entitled “Optical Switch to Enable Cell Sorting in a MicrofluidicChannel Network”. All of the above-referenced applications are hereinexpressly incorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to methods and apparatus for the use of opticalforces in a microfluidic channel network to provide an optical switchthat enables selective routing of target cells through the network tosort them from non-target cells and collect them.

BACKGROUND OF THE INVENTION

Conventional fluorescent activated cell sorters (FACS) are widely usedin research and clinical applications¹. These instruments are capable ofvery fast, multiparameter analysis and sorting but generally requirelarge sample volumes, a trained operator for operation and maintenance,and are difficult to sterilize. FACS instruments are able to analyze asfew as 10,000 and as many as tens of millions of cells. However, below100,000 cells the ability to perform sorting diminishes¹. Otherseparation methods such as magnetic beads don't require as many cells asFACS but they suffer from nonspecific binding, aggregation of cells andbeads, and from the possibility that the beads themselves couldinterfere in subsequent processing steps. Thus, for sorting precious,small samples or cells from primary tissue, a cell sorter that iscapable of handling small sample volumes with low cell numbers and thatallows efficient recovery of the sorted populations addresses a uniquescientific niche.

Microfabricated cytometers have the potential to sort with as few as1,000 cells while concomitantly consuming less reagents in an easy touse, closed system. The latter is important because, unlike conventionalFACS instruments, aerosols are not created, reducing the risks ofcontamination of the sorted cells and of working with biohazardousmaterials. Several microfabricated cell sorters have been described, butmostly as “proof of concept”. Fu, et al.² reported 30-fold enrichment ofE. coli at a throughput of 17 cells/s. Only 20% of the bacteria wereviable after sorting and the sort purity in the target reservoir was30%. In a subsequent study³, the throughput increased to 44 cells/s butthe target purity decreased to less than 10%, with recovery reported as39%. Wolff, et al.⁴ were able to sort beads from chicken red blood cellsat a throughput of 12,000 events/s, with 100-fold enrichment. However,purity in the target well was about 1%. In these studies, enrichment wasdefined as the increase in the concentration of the target population inthe collection well compared to the starting concentration. Purityreferred to the accuracy of the sort and was the percentage of targetcells sorted over all cells sorted into the collection well. Recoverywas defined as the number of cells counted by the fluorescent detectorvs. cells recovered from the collection well. The latter two studiesused pressure switches in microfluidic devices that switched the entirefluid flow path and, consequently, any particles contained within thefluid plug. The mechanical compliance in these switches caused the fluidswitch speed to be the rate limiting step for throughput³.Electrokinetic flow control has also been reported, e.g.,electroosmosis^(2,5,6) or dielectrophoresis^(7,8,9), but the highelectric field gradients and physicochemical restrictions on the ionicstrength of the buffer are non-ideal conditions for cells.

Buican et al.⁹ first proposed the use of optical forces for thedeflection of particles through a fluidic channel. The force exerted ona particle by an optical beam is a function of the optical power and therelative optical properties of the particle and its surrounding fluidmedium. Forces on the order of 1 pN/mW can be achieved for biologicalcells approximately 10 μm in diameter. While the optical force is small,the force necessary to deflect a cell into an adjacent flowstream isalso small, e.g. 900 pN to move a 10 μm diameter cell, 20-40 μmlaterally across the flow in a few milliseconds. This is the forcenecessary to overcome the viscous drag force on the cell at the velocityimplied by this lateral motion.

The principles behind the optical forces and general backgroundtechnology may be found in U.S. Pat. No. 6,744,038, which isincorporated herein by reference as if fully set forth herein.

SUMMARY OF THE INVENTION

As described below, these optical forces are used to realize an opticalswitch in a microfluidic channel network, operable as a cell sortingsystem. The optical switch is triggered by detection of a fluorescencesignal from target cells flowing in the microfluidic channel networkupstream of the optical switch position, although other detectionmodalities such as light scattering could equally be used for activationof the optical switch. The optical switch is used to direct cells orparticles into one of a multiple number of output channel flow streamswithout modifying the underlying flow, whereby the desired cells arecollected for further use. It is important that the flow in amicrofluidic channel is typically laminar at a very low Reynolds number.Consequently, any cell flowing in a particular lamina, or flow stream,will stay in that flow stream in the absence of any forces transverse tothe lamina. The optical switch utilizes optical forces on a cell toaccomplish just this, the transport of cells transverse to the lamina tomove the cells from a flow stream that exits a bifurcation junctionthrough one output channel to a flow stream that exits the bifurcationjunction through the second output channel.

The invention described in the following paragraphs details themethodology used to create an optical switch and the approaches used tooptimize the optical switch, the design of the microfluidic channelnetwork and the properties of the flow of cells or particles in themicrofluidic network in order to achieve enhanced sorting performance.The optical switch generally works by projecting an optical illuminationfield into the microfluidic channel network in the vicinity of thecell's trajectory in an established flow in a microfluidic channel. Theinteraction of the cell with the optical field produce forces on thecell that transport it transverse to the established flow such that itmoves from one flowstream to another flowstream in the established flow,without trapping the cell or significantly altering its motion in theprimary flow.

In the following text the terms cells and particles both will beunderstood to mean any of biological cells, biological particles,natural organic or inorganic particles and man-made organic or inorganicparticles. The size range of the cells sorted in the microfluidicchannel network is typically that of biological cells, with diametersranging from approximately 1 μm to approximately 50 μm. More generally,cells with diameters ranging from approximately 100 nm to approximately100 μm are candidates for sorting by means of an optical switch in amicrofluidic channel network.

Also, in general a laser has been used to produce the optical beam usedin the optical switch. The laser currently used for the optical switchis a near-IR, continuous wave laser that is known not to harm theviability of biological cells at the power densities and exposure timesused to demonstrate optical switching. Alternate laser sources may beconsidered for different applications, including visible or near-UVwavelength lasers if damage to the particles is not an issue, or pulsedlasers where a large flux of light can be used to move the particle veryquickly. However, the source of the optical beam does not need to belimited to a laser, even though further discussion of the invention usesa laser to produce the optical switch.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a “Y” shaped sorting junction in a microfluidicchannel network.

FIG. 2 is a plan view of a microfluidic channel network thatincorporates both a sheath flow pinch junction and a “Y” shaped sortingjunction connected by a main channel, with 50/50 splitting of cells inthe flow, collectively referred to as a 50/50 optical switch network.

FIG. 3 is a plan view of a microfluidic channel network thatincorporates both a sheath flow pinch junction and a “Y” shaped sortingjunction connected by a main channel, with skewed splitting of cells inthe flow via differential sheath flow, collectively referred to as asheath flow skewed optical switch network, with an optical switch.

FIG. 4 is a plan view of a microfluidic channel network thatincorporates both a sheath flow pinch junction and a “Y” shaped sortingjunction connected by a main channel, with skewed splitting of cells inthe flow via differential outlet channel width, collectively referred toas an outlet flow skewed optical switch network, with an optical switch.

[Note that in subsequent figures text based labeling has been usedinstead number based labeling; nevertheless, in similar figures thenumber based labeling of FIGS. 1-4 still applies.]

FIG. 5 is a 50/50 optical switch network with a bi-directional laserline optical switch.

FIG. 6 is a 50/50 optical switch network with a bi-directional laserspot optical switch.

FIG. 7 is a plan view of laser line optical switches in largermicrofluidic channel networks with more than two outlet channels.

FIG. 8 shows possible optical designs for modulation and/or shutteringof the optical switch.

FIG. 9 is a plan view of a sheath flow skewed optical switch networkwith a laser spot optical switch that is translated parallel to the cellflow or at an angle to the cell flow.

FIG. 10 shows a detector arrangement and timing/trigger diagram using asingle laser source for the cell detection and trigger decision method.

FIG. 11 shows a detector arrangement and timing/trigger diagram usingtwo laser sources for the cell detection and trigger decision method.

FIG. 12 is a schematic of a representative design for photolithographymasks for microfluidic channel networks in both bottom and top glasssubstrates that provides a 2-dimensional sheath flow pinch of the cellflow in the main channel when these substrates are bonded to form asingle network.

FIG. 13 shows a 3-dimensional illustration of the design described inFIG. 12.

FIG. 14 is an illustration of the side view of a microfluidic channelnetwork that provides sequential sheath flow pinch of the cell flow inthe vertical direction and then in the horizontal direction, resultingin full 2-dimensional sheath flow pinch of the cell flow in the mainchannel.

FIG. 15 is a 3-dimensional illustration of the microfluidic channelnetwork described in FIG. 14.

FIG. 16 is a schematic of a representative photolithography mask designfor both bottom and top glass substrates that when bonded together formthe microfluidic channel network illustrated in FIGS. 14 and 15.

FIG. 17 is a representative embodiment of a photolithography mask for acomplete microfluidic channel network, with a T-pinch junction and aT-bifurcation junction to the outlet channels, to implement the opticalswitch based cell sort method.

FIG. 18 is a representative embodiment of a photolithography mask for acomplete microfluidic channel network, with a triangle-pinch junctionand a Y-bifurcation junction to the outlet channels, to implement theoptical switch based cell sort method.

FIG. 19 shows a preferred embodiment of a microfluidic channel networkin a completed microfluidic cell sorting chip.

FIG. 20 shows a preferred embodiment for a self-contained disposablecartridge for the optical switch based microfluidic channel network cellsorter.

FIG. 21 shows a preferred embodiment of the optical system for theoptical switch based microfluidic channel network cell sorter.

FIG. 22 shows representative performance of the optical switch basedmicrofluidic channel network cell sorter for various implementations ofthe optical switch.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows one embodiment of an optical switch 10 that serves to sortcells in a 1×2 microfluidic channel network, i.e. a network with onemain input channel 11 and two output channels 12 and 13 extending from abifurcation junction. A “Y” geometry for the bifurcation junction isshown in FIG. 1, but other bifurcations such as a “T” geometry may alsobe used. In general these microfluidic channels are produced inoptically transparent substrates to enable projection of the opticalswitch and other cell detection optics into the channel. This substrateis typically, but not limited to, glass, quartz, plastics, e.g.,polymethylmethacrylate (PMMA), etc., and other castable or workablepolymers (e.g. polydimethylsiloxane, PDMS or SU8). The depth of themicrofluidic channels is typically in, but not limited to, the range 10μm to 100 μm. The width of the microfluidic channels is typically, butnot limited to, 1 to 5 times the depth. The cross section is typicallyrectangular, or rectangular with quarter-round corners in the case ofmicrofluidic channels produced by photolithograpic masking of glasssubstrate followed by isotropic etching of the channels.

The flow conditions are set such that when the optical beam, in thiscase from a laser, is turned off or blocked so that the beam does notimpinge on the junction region, all cells will preferentially flow intoone of the output channels, for example the right output channel 13.When the optical beam is turned on or unblocked, the beam strikes thejunction region and optical forces generated by the interaction of thecells with the optical beam direct the cells into the left outputchannel 12. In this example, the optical pattern chosen for directingthe cells is a long, thin line of laser illumination at some anglerelative to the direction of fluid flow. Optical gradient forcesdisplace the cells laterally, away from the main stream line of cells,such that switched cells then exit the main channel into one outputchannel, for example 12 while unswitched cells from the main stream ofcells exit into the other output channel, for example 13. The settingand control of the flow conditions in the microfluidic channel networkcan be achieved by direct drive pumping, pneumatic pumping,electro-kinetics, capillary action, gravity, or other means to generatefluidic flow.

The performance of the sorting mechanism in terms of throughput (thetemporal rate of cells entering the sorting region at the top of thebifurcation junction), yield efficiency (the fraction of target cells inthe target output channel, 12), and purity (the ratio of the number oftarget cells to the total number of cells in the target output channel,12), are impacted by various factors, each of which affects theimplementation of the optical switch. The optical switch can becharacterized by several parameters such as the shape of the opticalpattern projected into the sorting junction region of the microfluidicchannel network, the position of the pattern with respect to thebifurcation junction, any motion of the optical pattern with respect toits initial position and shape, the duration of activation of theoptical switch, the wavelength and power of the laser source used toproduce the optical switch pattern, etc. The selection of particularvalues of these parameters for the optical switch is a critical functionof, among other things, the topology and geometry of the microfluidicchannel system, the flow rates (cell velocities) within the microchannelsystem, the ability to control the position of cells flowing in the mainchannel (whether they are flowing in the center of the main channel oroff-set to one side), the amount of displacement of the cells necessaryto achieve reliable switching, the depth of the channels, the shape ofthe channels, and the forces produced by the cells' interactions withthe optical switch.

In general, when cells are introduced into the flow in the main channelthey may move down the channel at any transverse position within theflow. Consequently the cells may be moving at differing velocities,depending upon their transverse positions due to the well knownparabolic (for cylindrical microfluidic channels) or quasi-parabolic(for more general cross sections) velocity profile of pressure drivenflow in microfluidic channels. This would make it difficult to bias theflow of all cells to one output channel, say 13, as shown in FIG. 1. Anyimplementation of an optical switch with this flow geometry wouldnecessarily result in low throughput and inefficient use of the laserpower available to produce the optical switch. The use of appropriateflow conditions can help alleviate these restrictions on the performanceof the optical switch.

Establishing the appropriate flow conditions can be done in many ways.In one embodiment, 1-dimensional focusing of cells (horizontally in theplanar view shown) into a single file in the center of the main channelis achieved by pinching the cell input channel flow 20 with added flowof buffer from both the left 21 and right 22 sides, using a sheath flowapproach as shown in FIG. 2. Maintaining the cells in the center of themain channel is achieved by having equal flow from each side. This floweffectively creates a fluidic splitting plane 23, as shown in FIG. 2,and this ultimately will result in a 50/50 splitting of the fluid andcells at the bifurcation junction. Implementation of an optical switchto sort target cells from a mixed population of cells using thismicrofluidic channel design and flow conditions requires an opticalswitch that actively switches both target cells to one output channel,say 12 as shown in FIG. 1 and non-target cells to the other outputchannel, say 13.

Alternatively, the focused line of cells can be positioned off-set fromthe center of the main channel by putting unequal flows into the sidesheath flow channels, FIG. 3 a-b. This effectively causes a skewed flowof cells from the input channel 30 to one side of the splitting plane 33within the main channel. The side of the main channel to which the cellflow is skewed will be opposite to the side in which the sheath flow hasthe higher flow rate. That is, when the right sheath buffer 32 flowsfaster than the left sheath buffer 31, the line of cells is skewedtoward the left of the flow in the main channel, as shown in FIG. 3 a-b.However, the left sheath flow could also have the higher flow whichwould push the line of cells toward the right side of the main channel.Also shown in FIG. 3 a-b are a fluorescence detector 34 and an opticalswitch 35. The fluorescence detector is used as a means to decide whichcells to sort, and will be discussed in further detail later. It isevident from FIG. 3 b that an effective sort involves moving a cellacross the splitting plane from a flow stream that exits the bifurcationjunction to the fluorescence-negative non-target cell microfluidicchannel 36 into a flow stream that exits the bifurcation junction to thefluorescence-positive, target cell microfluidic channel 37. Manipulationof the sheath buffer flow rate can be achieved either by separatelycontrolling the flow rate in the respective side channels using directdrive pumping, pneumatic pumping, electro-kinetics, capillary action,gravity, or other means to generate fluidic flow, or by specificallydesigning the microfluidic sheath network to ensure that central flow(50/50 splitting) or off-set flow occurs, through careful balancing ofthe pressure drops in each of the microfluidic channels.

An alternative approach to achieve the preferential flow of all cellsfrom the input flow 40 in the main channel into one output microfluidicchannel, say the fluorescence-negative channel 46, prior to fluorescencedetection 44, is to obtain central pinching using equal sheath bufferflow rates 41 and 42, but then preferentially bias the cell flow intofluorescence-negative channel by having a larger volumetric fluid flowout of the bifurcation junction into the fluorescence-negative outputchannel 46 relative to the fluorescence-positive output channel 47. Thisis demonstrated in FIG. 4 a-b, in which the left output channel 46 iswider than the right output channel 47. This configuration effectivelyplaces the splitting plane 43 to the right of the centrally located cellstream. Therefore, with the cells in the desired position, the opticalswitch 45 is then used to translate the target cells across thesplitting plane into the target cell, fluorescence-positive, rightoutput channel. This approach is equally effective by having the rightoutput channel wider than the left output channel, whereby target cellsare translated by the optical switch across the splitting plane, whichis now located to the left of the centrally located cell stream, and areconsequently sorted into the left output channel. Thus, by specificallydesigning the microfluidic channel outlet network, or by activelycontrolling the outlet back pressure in the respective outlet channels,the flow of cells into a desired output channel can be controlled.

The use of either central flow or an off-set flow, and the respectivedistance of the focused cell flow from a fluidic splitting plane,ultimately dictates the magnitude of the displacement of the cellsnecessary to achieve reliable switching. This further dictates thelength of the laser line and the laser power required to achievereliable switching. The closer the cell stream is to the splittingplane, the shorter the displacement required, and the more efficient thesorting process becomes. For enhanced purity of the sorted populationand for high throughput, the single optical switch in a mono-directionalarrangement requires the sample stream be offset from the splittingplane. In this manner the occurrence of a mistaken sort is minimized.

An alternative to this design is to use a bi-directional optical switchwhich utilizes two laser lines. With this approach one laser line sortsthe desired cells to one output channel, and the other laser line sortsall other cells into the other output channel. This approach can be usedwith either the 50/50, FIG. 2, or the offset, FIGS. 3 and 4, splittingconfiguration. In the latter case when a cell is not in the switchingzone, one may choose to leave the laser on in either of its twopositional states, or one may also shutter the laser during this time.The optical switch can also be made bi-directional by having twomirror-image laser lines impinging on the switching region, located justabove the bifurcation junction, which independently turn on to directcells to either of the two outputs stemming from the bifurcationjunction.

A schematic of the bi-directional optical switch using laser lines in a1×2 microfluidic network is shown in FIG. 5. A similar bi-directionaloptical switch has also be achieved with laser spots directed to eitherside of the channel, as shown in FIG. 6. As with the mono-directionaloptical switch, a single laser source can be used in the bi-directionaloptical switch, or alternatively the bi-directional optical switch canuse two independent laser sources. The bi-directional design potentiallyoffers some performance advantages versus the mono-directional design.The first is that purity is potentially maximized because every cell isdirected by the laser. Secondly, the fluid flow is simplified becauseequal flow can be directed out each of the two output ports, instead ofsome predetermined ratio of flow.

Although only the 1×2 microfluidic channel design with flow through oneinput main channel into a bifurcation to two output channels has beenconsidered thus far in this description, microfluidic networks with 1×N,or M×N, outputs can be utilized. Optical switching can be achieved inthese larger networks by having an arbitrarily large number ofindependently modulated laser lines. Some embodiments are shown in FIG.7 a-c. Furthermore, cells can also be fed back multiple times throughthe same sorter to increase the purity of the sort, or alternatively,channels can also be arranged in a cascade for multiple levels ofsorting.

Two different activation modes can be considered when operating theoptical switch in a mono-directional or bi-directional arrangement; apassive mode or an active mode. The passive mode is such that the stateof the optical switch is either on or off, regardless of what cell maybe flowing through the channel. In this case knowledge of when or howmany cells are entering the switching region is not required, andconsequently, depending on the state of the laser, all cells within theswitching region are switched. Alternatively, in the active mode thecells are first detected as they enter a detection/selection region, andthen are switched based on some decision process. FIG. 3 a-b and FIG. 4a-b show examples of this mode that use a fluorescence detector placedjust prior to the switching region. In this case, all fluorescent cellswere directed to one output channel, and all non-fluorescent cells weredirected to the other output channel. Other non-fluorescentdetection/selection techniques for the decision process includeTime-Of-Flight, scatter, imaging, capacitance, or any detection modalitythat can identify a desired cell. Regardless of the detection/selectionmethod, switching using the active mode can be utilized to sort onepopulation of cells from another based on some decision process.

In order to utilize the active mode, the optical beam must be modulatedon or off in response to the decision process. Regardless of the numberof lasers used, or whether the optical switch is mono-directional orbi-directional, the lasers can be modulated in many ways, includingusing an electro-optic modulator, modulating the laser power, shutteringthe laser, using a liquid crystal modulator, using a galvanometer, andusing an acousto-optic modulator. For the bi-directional optical switchwith two lasers, the separate lasers can be turned on and offindependently; however, when using a single laser source the twodifferent orientations of the optical switch line can be achieved byusing a polarization rotator (such as a liquid crystal modulator) andhaving each of the two different line patterns be each of two separatepolarizations. Similarly, an acoustic-optic modulator or a galvanometermirror can be used to modulate the position of a single spot used as theoptical switch, or a two-axis acousto-optic modulator or two-axisgalvanometer mirror can be used to draw two different line shapes to beused as the bi-directional optical switch.

FIG. 8 shows three different possible optical designs for performing themodulation and/or shuttering of the optical switch. In FIG. 8 a thebi-directional optical switch is created from a single optical beam(laser) directed toward and passing through a Liquid Crystal Modulator(LCM). The LCM is a polarization rotator and therefore if the beam ispolarized in one direction it will pass straight through the PolarizingBeam Splitter (PBS), through a cylindrical lens creating a line shape,through another PBS, and then through some focusing optics which focusthe line onto the microfluidic switching region. This effectivelycreates one line of the bi-directional optical switch used to switchcells into one of the bifurcated channel outputs. To switch cells intothe other output channel a mirror image line must be created. This isaccomplished by rotating the LCM which alters the polarization of thebeam. Consequently, when the beam strikes the first PBS it is directedinto an alternate path through a different cylindrical lens (creating aline shape), through the other PBS, which directs the beam back throughthe focusing optics which focus the mirror image line onto themicrofluidic switching region. Note that the cylindrical lenses wereused to create the line shape for the bi-directional optical switch;alternatively the cylindrical lenses can be removed resulting in spotsused for the optical switch. In FIG. 8 b, rather than use thecombination of the LCM and PBS, with or without the cylindrical lenses,an Acousto-Optic Modulator (AOM) can be used to create the lines orspots used in the bi-directional optical switch. This is achieved byconfiguring the AOM to obtain the desired line shape that is required.Also, the AOM can be used to shutter the optical beam in an on/offfashion, directing the beam to a beam stop for the Optical Switch offcondition. FIG. 8 c shows the combination of the systems described inFIG. 8 a and FIG. 8 b. In any configuration that uses an AOM to vary thedirection of the beam, a galvanometer mirror, either one-axis ortwo-axis, depending on the desired beam motion, may be used in place ofthe AOM.

Many variations for the optical pattern can be considered whenoptimizing switching efficiency for mono- or bi-directional opticalswitches. As mentioned above a laser line has been used as the opticalswitch pattern. The line might be generated by a cylindrical lens, byscanning a galvanometer mirror or an acousto-optic modulator, by adiffractive optic, by a custom refractive optic, or by any othertechnique. To date the line has been generated using a cylindrical lens,by scanning a galvanometer or by using an acousto-optic modulator. Thelength of the line can be arbitrarily long or as short as a singlepoint. The line can have higher intensity at the top of the line andgradually taper down in intensity toward the end of the line.Additionally the line might be a curved arc which optimizes the outputdirection of the cells. Additionally, in real time the angle of the lineor the shape of the line might vary (i.e. swivel to optimize output).For implementations with multiple output channels, any arbitrary patternof lines in 2D space might be generated to optimize the direction ofeach output cell. Alternatively, the line might be created by an arrayof discrete spots.

To further improve the performance of the sorting mechanism in terms ofthroughput, yield efficiency, and purity, the optical switch has beenconfigured such that the laser spot is swept alongside a selected cellas it flows down the main channel toward the bifurcation junction,thereby increasing the total interaction time between the cell and thelaser. The optical switch utilizes a laser spot which is translated, ina straight line, down the length of the main channel toward thebifurcation junction. The line swept by the spot can be parallel withthe walls of the main channel (FIG. 9 a), or can be at some anglerelative to the cell flow stream (FIG. 9 b). Therefore, the angle canrange from 0-90 degrees. The ability to sweep the spot is achieved usingeither an AOM or scanning galvanometer mirrors. The optical switch istriggered to sweep by a decision based on detection of the desired cellusing fluorescence or other detection modality that can identify adesired cell; for example Time-Of-Flight, scatter, imaging, orcapacitance. The cell position can be either off-set or centered in themain channel, which dictates the length of the line swept by the spotand the laser power used to achieve efficient switching/sorting. Thus,as a desired cell is detected the optical switch is turned on, and thespot appears alongside the desired cell. The spot then tracks alongsidethe selected cell and uses optical forces to direct the selected cellinto the desired output channel.

Two approaches to facilitate efficient triggering of the optical switchare described below. Typical to both methods is the use of a temporalsignal to analyze the moving cell, and use this information to generatea decision to switch, or not to switch. This temporal signal isessentially a measure of a signal as a function of time, which can yielda distinctive temporal fingerprint in terms of both peak intensity andpeak width. The signal may be fluorescence, scatter (for instance,forward scatter), capacitance, imaging, or any detection modality thatcan identify a desired cell. One approach is to utilize a single lasersource coupled with two or more detectors to accomplish both celldetection and cell identification. FIG. 10 a-d show this approach usingone laser source combined with a fluorescence detector and a forwardscatter detector. The temporal signals from these detectors are used asthe information for the switch decision. The presence of a cell isverified by the forward scatter signal and when this signal is coupledwith a fluorescence signal intensity which is within a predeterminedrange; this “gating” information is then used to trigger the opticalswitch. Note that only a single fluorescence detector is shown, howevermultiple fluorescence detectors can be used for further refined cellidentification. In the case depicted the cell stream is centrallylocated by using equal flow rate sheath buffers, with output channelshaving different widths used to create a splitting plane to the right ofthe cell stream. However, any configuration used to manipulate theposition of the cell stream and splitting plane, as discussed above, canbe used. Also, common to both configurations is the presence of an errorchecking detector, which verifies whether a cell has been switched ornot. The detection in this case can be based on fluorescence, scatter(for instance forward scatter), capacitance, imaging, or any detectionmodality that can identify a desired cell.

FIG. 10 a-b show the detector arrangement and the timing/trigger diagramfor when the sort parameter is negative and the optical switch is nottriggered. The cells enter the main fluidic channel and are focused intoa single file by sheath buffer flowing from both sides. As a cell passesthe through the laser in the detection/selection region, bothfluorescence and forward scatter signals are detected simultaneously, ornearly simultaneously. Although the presence of a cell is successfullydetected via the forward scatter signal (at time t₁), the fluorescencesignal is below the gating level and the optical switch is not triggered(at time t₂). Thus, no error check signal (at time t₃) is obtained sinceno cell was switched. Alternatively, FIG. 10 c-d show the detectorarrangement and the timing/trigger diagram for when the sort parameteris positive and the optical switch is triggered. Here, as a cell passesthrough the laser in the detection/selection region, both fluorescenceand forward scatter signals are again detected (at time t₁)simultaneously, or nearly simultaneously, but the fluorescence signal iswithin the gating level and the optical switch is triggered (at timet₂). An error check signal (at time t₃) is obtained since a cell wasswitched. In this approach the trigger time (at time t₂) is a presetvalue (Δt) measured from the initial detection time (t₁), and this Δtvalue is determined by the speed of the cells and the position of theoptical switch relative to the detection/selection region. This approachis satisfactory to achieve efficient sorting; however as a means tofurther improve the triggering accuracy a second approach is used.

FIG. 11 a-d shows this second approach, in which two laser sources areused instead of one. Also, as with the single laser approach describedabove, the temporal signals from these detectors are used as theinformation for the switch decision. One laser is used in a detectionzone to separately accomplish cell detection prior to theidentification/selection region. The detection in this case can be basedon fluorescence, scatter (for instance forward scatter), capacitance,imaging, or any detection modality that can identify a desired cell. Thesecond laser is coupled with two or more detectors and is used toaccomplish cell detection and cell identification. Again, identificationin this case can be based on fluorescence, scatter (for instance forwardscatter), capacitance, imaging, or any detection modality that canidentify a desired cell. The purpose for two sequential cell detectionsteps is such that the cell flow rate can be obtained from the timedifference (Δt) between the first detection (at time t₁) and the seconddetection (at time t₂). Knowing the spacing between detector windows (d)will yield the flow rate (v=d/Δt), and this value combined with theknown distance the optical switch is from the identification window (x)is then used to calculate the triggering time for the optical switch(t₃=x/v). Again switching only occurs when specific gating levels arereached for the cell identification step. Although only a singlefluorescence detector is shown for identification, multiple fluorescencedetectors can be used. In the case depicted the cell stream is centrallylocated by using equal flow rate sheath buffers, with output channelshaving different widths used to create a splitting plane to the right ofthe cell stream. However, any configuration used to manipulate theposition of the cell stream and splitting plane, as discussed above, canbe used. Also, common to both configurations is the presence of an errorchecking detector, which verifies whether a cell has been switched ornot. The detection in this case can be based on fluorescence, scatter(for instance forward scatter), capacitance, imaging, or any detectionmodality that can identify a desired cell.

FIG. 11 a-b shows the detector arrangement and the timing/triggerdiagram for when the sort parameter is negative and the optical switchis not triggered. The cells enter the main fluidic channel and arefocused into a single file by sheath buffer flowing from both sides. Thepresence of a cell is verified by the forward scatter signal (at timet₁) as it passes through the detection window region. As the cell passesthrough the identification/selection window a second forward scattersignal is obtained (at time t₂), however, this signal is coupled with afluorescence signal intensity (at time t₂) which is not within thegating level and the optical switch is not triggered (at time t₃). Noerror check signal (at time t₄) is obtained since no cell was switched.Even without sorting a cell the flow rate (v) of the cell stream isobtained using (t₁), (t₂) and the known distance (d) between thedetection and identification windows. This is obtained using therelationships: Δt=(t₂)−(t₁) and v=d/Δt.

Alternatively, FIG. 11 c-d show the detector arrangement and thetiming/trigger diagram for when the sort parameter is positive and theoptical switch is triggered. Here, the presence of a cell is againverified by the forward scatter signal (at time t₁) as it passes throughthe detection window region. As the cell passes through theidentification/selection window a second forward scatter signal isobtained (at time t₂), and this signal is coupled with a fluorescencesignal intensity (at time t₂) which is within the gating level and theoptical switch is triggered (at time t₃). An error check signal (at timet₄) is now obtained since a cell was switched. In this approach thetrigger time (t₃) is not a preset value, but rather it is calculatedusing the cell stream flow rate (v) and the known distance (x) betweenthe optical switch and the identification window. This is obtained usingthe relationships: Δt=(t₂)−(t₁); v=d/Δt; (t₃)=x/v. This approach allowsfor more efficient sorting as it can account for fluctuations in cellflow rate, and therefore more accurately trigger the optical switch. Anadded benefit of this approach is, for each individual cell, thepossibility of adjusting the rate at which the laser spot is translateddown the channel such that it matches the velocity of the cell asdetermined above, thus maximizing the interaction time between the celland the laser spot of the optical switch. The translation velocity ofthe laser spot would be varied by varying the driver for the AOM.

Another approach to improving the sorting efficiency, whileincorporating the triggering approaches described above, is tocentralize the cells in the main channel using channel designs whichcreate a true sample core, whereby the core is completely surrounded bythe sheath buffer. Variability in the location of a cell along thechannel height can cause variability in cell detection and fluorescenceintensity. Ensuring that the cells are in a core flowing in the centerof the main channel may improve sorting efficiency, since this minimizesany variability due to radial distribution of cells, and controls thedistance the cell needs to be moved to effect efficient sorting. Such acore flow can be achieved with a 2-dimensional pinch of the input flowstream with sheath buffer.

This approach requires a bottom substrate and a top substrate; each withmicrofluidic channel networks formed in them. FIG. 12 a-b and FIG. 13show one method to accomplish this, in which the channel design on onesubstrate is the mirror image of the design on the other substrate.Thus, when the two substrates are brought together, with the channeldesigns facing each other, the channel networks overlay and formcomplete fluidic conduits. FIG. 12 a-b show one type of design used inthis approach, with the sample channel shown as a dashed line. The keyfeature of this approach is to ensure that the sample channels areshallower than the sheath channels, such that when the substrates arebrought together the sample conduit appears to enter the junction as ahole. This is shown in FIG. 13, where the cells can be seen to enter thejunction, and then are pinched from all sides creating a sample corewhich flows in the center of the main channel. Note that the channelscan be formed by wet chemical etch or laser etch of glass or quartz, bymolding or embossing in plastics or polymers.

Another method involves having a series of intersecting channelsarranged such that in the first junction/intersection the cells arepushed vertically toward one wall of the main channel, the nextjunction/intersection forces this cell stream vertically into the centerof the main channel, and then a final pinch flow from both sides at athird junction/intersection creates the complete sheath buffer shroudaround a sample core flowing in the main channel. This is shown in FIG.14 and FIG. 15, with one possible channel schematic shown in FIG. 16. Inthis example, at junction (A) sample flows from the top substrate intothe junction and down into the channel in the bottom substrate, wherethe side sheath buffer flows into the junction from the sides. Thesample is slightly focused and pushed to the top wall of the bottomchannel as it continues to flow toward the next junction (B). Atjunction (B) the sample flows along the top of the bottom channel fromjunction A to junction (B). Here a second sheath buffer flows into thejunction (B) from the top substrate and the sample is pushed down to themiddle of the channel in the bottom substrate. The sample continues toflow along the middle of the bottom channel toward the next junction(C). Here a third sheath buffer flows into junction (C) from both sides,and the sample is pinched into single file. The sample is now surroundedby sheath buffer as it continues to flow, as a sample core, centeredboth horizontally and vertically within the main input channel.

All of the microfluidic channel network designs described in FIGS. 1-16have been produced in glass substrate utilizing conventionalphotolithographic masking and isotropic etching of the masked glasssubstrates. The isotropic etch typically produces microfluidic channelsthat have a depth d_(e) at the center of the channel and a widthw=w_(p)+2×d_(e) at the top of the channel, where w_(p) is the width ofthe photolithography pattern that defines the channel. The bottomprofile of the channel has a quarter-round contour of radius d_(e) ateach edge due to the isotropic etch and the top of the etched channel isopen. A glass substrate, typically a glass cover slip, is thermallybonded to the substrate with the etched microfluidic channels to sealthe tops of the channels and complete a microfluidic channel network.Holes are typically drilled in the top substrate prior to the thermalbonding to provide vias for ingress and egress of fluid flow to themicrofluidic channel network. The depth d_(e) of the channels depends onthe rate of the chemical etch process and the duration of the etch step.The depth of the microfluidic channels is typically in, but not limitedto, the range 10 μm to 100 μm. The width of the microfluidic channels istypically, but not limited to, 2 to 5 times the depth. This is achievedby using lines on the photolithography mask that are typically, but notlimited to, the range 5 μm to 400 μm. As mentioned previously, othersubstrates may be used, such as plastics or moldable or castablepolymers. In these cases, the microfluidic channels typically haverectangular cross sections, but otherwise are similar to the channels inthe glass substrates. The size of the glass substrate in which themicrofluidic channel network is produced is typically in, but notlimited to, the range of 5 mm×5 mm to 25 mm×50 mm with a total thicknessin, but not limited to, the range 500 μM to 2 mm. The top substrate istypically the same size, with thickness in, but not limited to, therange 300 μm to 1 mm. The vias are typically, but not limited to, 200 μmto 600 μm in diameter. The completed substrate, with a microfluidicchannel network and a bonded cover plate with vias for fluidic ports foringress and egress of fluid flow, is termed a microfluidic sorting chipor chip for brevity.

The microfluidic channels networks shown in FIGS. 1-16 typically haveonly described the local geometries of the inlet microfluidic channel,the sheath buffer pinch junction channels, the cell identification andoptical switch main channel, and the bifurcation of the main channel tothe outlet channel. This description needs to be expanded to provide forregions in each channel to make the connections to reservoirs in amacro-scale fluidic device or cartridge that provides the interface tothe vias described above to provide ingress and egress of the fluid flowfrom the network. The cross section and length of each of thesemicrofluidic channels typically needs to be adjusted to assureappropriate controlled flow within the entire microfluidic channelnetwork, depending on the technique selected to achieve the flow in thechannels. Both the cross section and the length of these channels aredetermined by the pattern used to produce the photolithography mask.

FIG. 17 shows one embodiment of a mask for a complete microfluidicchannel network that has an inlet channel, two sheath channels to aT-pinch junction and two outlet channels from a T-bifurcation junction.This mask was designed to provide a 7:1 volumetric pinch ratio (thesheath flow rate is seven times greater than the cell inlet flow rate).The length of the channels was designed to provide both sufficientpressure drop to enable the use of either standard low flow syringepumps or low pressure pneumatic controllers to establish the flow. Thedesign also reflects the balance of pressures needed to enable use ofonly two pumps, one for the cell inlet channel and one for the twosheath channels, with the outlets maintained at atmospheric pressure.The sheath channel inlet is at the termination at the top of the design,the cell inlet channel originates below this in the center of the twosheath channels and is long enough to provide the appropriate pressuredrop to set the 7:1 pinch ratio, and the two outlets are located at thetermini at the bottom left and right.

FIG. 18 shows another embodiment that incorporates a triangular junctionfor the pinch junction and a Y-bifurcation junction, in a design thatprovides a 10:1 volumetric pinch ratio. Otherwise the design isgeometrically similar to that of FIG. 17. Many other designs are clearlypossible, but they all share the common features of needing to providefor fluidic ingress and egress and to provide appropriate pressure dropsand pressure balances for the method chosen to establish the fluid flow.Similar design conditions are used to produce the photolithography masksused to make the microfluidic channel networks for 2-dimensional pinchflow networks described previously.

FIG. 19 shows a preferred embodiment of a microfluidic channel networkin a completed microfluidic sorting chip. The two inlet ports, for thecell sample flow and for the sheath buffer flow are identified, as arethe two outlet ports, for the fluorescence-positive target cells and forthe fluorescence-negative non-target cells, the waste stream. The chipis 24 mm by 40 mm. The thickness of the etched substrate is 1.1 mm. Thethickness of the bonded cover plate is 550 μm. The microfluidic channelsare 50 μm deep. The cell inlet microfluidic channel is 110 μm wide. Thesheath flow and outlet microfluidic channels are 150 μm wide, as is themain microfluidic channel. The sheath flow pinch junction is an invertedequilateral triangle, 300 μm per side, connecting the cell inlet channelthrough the base of the triangle, at the top of the junction, with thetwo sheath flow pinch channels from each side to the main channelthrough the apex of the triangle, at the bottom of the junction. Thismicrofluidic channel network design is optimized to use pneumaticcontrol of the flow at all four ports to establish the network flow.

Microfluidic connections to the chip may be made in a variety of ways.One method is to use flexible microfluidic tubing directly connected tothe ports, either by gluing or using various tubing adapters that can beattached to the surface of the chip at the ports. This tubing can beconnected directly to syringe pumps or similar systems that providevolumes for handling both the cell sample and the sheath buffers andprovide the pressure to flow these volumes through the chip. Using thesyringe pumps for handling the sample volume requires that the pump becleaned and reloaded for each sample and introduces the possibility forcarry over or contamination from one sample to the next.

An improved method for microfluidic connections to the chip utilizes acartridge that is directly adhered to the chip using a UV-curableadhesive, a PSA bonding sheet, or other conventional bonding methods.The cartridge has four built-in reservoirs that separately provideinterface connections to the cell inlet channel, the two sheath channels(from one reservoir), and each of the two outlet channels. Such acartridge provides the possibility of sterile handling of both the cellsample and the sorted target cells and waste stream, since they can becompletely confined to the volumes of the cartridge before and after thecell sort. The flow for such a cartridge and chip system can be providedby using two pneumatic/pressure controllers that separately pressurizethe cell inlet and sheath buffer reservoirs to induce flow through themicrofluidic channel network of the chip to the outlet reservoirs thatare at atmospheric pressure.

An improved flow control method is provided by using four pneumaticcontrollers that separately pressurize each of the cell inlet, sheathbuffer, target cell collection and waste collection reservoirs. Such aflow control system provides the ability to separately adjust thevolumetric pinch ratio at the sheath pinch junction, the flow velocityof the cells in the main microfluidic channel for the fluorescenceanalysis and optical switch, and the split ratio at the switchingbifurcation to enable biased flow, as described previously.

FIG. 20 shows a preferred embodiment of a self-contained disposablecartridge 210 that provides fluidic reservoirs for the cell samplevolume 200, the sheath buffer volume 201 and the two outlet collectionvolumes 202 for target cells and waste respectively. The cartridge 210is manufactured from acrylic plastic and may either be machined or cast.Other plastics or suitable materials may be substituted for acrylic ifappropriate. The cell sample volume 200 is typically conical in shape,tapering towards the port to the inlet microfluidic channel 191. In thepreferred embodiment, the inlet reservoir contains a polypropyleneinsert to minimize cell adhesion and consequently maximize cell yield.The chip is bonded with UV adhesive to the optical window region 203,and the outlet ports from the chip interface with their respectivereservoir volumes. The reservoir volumes 200, 201, 202 are sealed withthe snap-on lid 204 that has drilled ports for connection between thepneumatic controllers and the individual reservoirs 200,201,202. The lid204 contains a silicone gasket to aid in sealing against the cartridgebody 210. It also incorporates a 0.1 μm polypropylene filter to create agas permeable, liquid tight interface between the cartridge volumes andthe external environment. This maintains aseptic conditions on thecartridge and minimizes any biohazard contamination to the user or theinstrument.

The cartridge 210 is prepared for a cell sorting run by first primingthe microfluidic channel network through the sheath port with sheathbuffer solution, using an ordinary syringe with a luer fitting. In thisway the channels are primed and the sheath reservoir 201 is filled with800 μl and each outlet reservoir 202 is filled with 200 μl. The cellsample reservoir 200 is aspirated of excess buffer liquid and then 5-25μl of cell sample is placed into the sample input reservoir 200 using apipette. The cartridge lid 204 is then applied and snapped into place,providing a self-contained system in which to perform the cell sortingrun.

The cartridge 210 is designed to be placed in a holder that positionsthe main channel 190 of the chip such that the optical imaging systemthat projects the optical switch beam into the channel is appropriatelyaligned and focused into the channel 190. The cartridge holder alsoincludes a pressure manifold plate that has four ports, connected byexternal tubing to the four pneumatic controllers 211. Each manifoldport is sealed to its respective cartridge lid port with an o-ring, andthese seals are made leak free by pressing the manifold against thecartridge lid with a cam-lock mechanism.

A preferred embodiment of the optical system for the optical switch isshown in FIG. 21. The cartridge 210, with the pneumatic manifold 211connecting to the snap-on lid 204, is positioned such that the opticalswitch region is at the focus of both a lens system viewing from above212, 215 the cartridge and a lens system viewing from below 213, 221.The output beam from a 488 nm laser 214 is projected through the imagingsystem 212,215 into the main channel just upstream of the sortingregion, as shown in FIGS. 3-7 and 9-11, to provide excitation for thedetection of fluorescence from fluorescence-positive target cells. Thefluorescence emission is collected by the same lens and imaged through adichroic mirror and an appropriate fluorescence emission filter to aphotomultiplier tube. The signal from the photomultiplier tube 217 isprocessed by the electronics to measure the level of the fluorescencefrom the cells and determine the presence of fluorescence-positivetarget cells in the flow stream in the main channel. The fluorescenceexcitation is not limited to the 488 nm wavelength, but can be at anywavelength that is appropriate for the fluorophores used to identify thetarget cells. If a different excitation illumination is used, thewavelength of the fluorescence emission filter must be changedaccordingly. When fluorescence-positive cells are identified, theelectronics triggers the AOM 219 to direct the beam from the IR-laser220, typically a 1070 nm laser operation between 5 W and 20 W outputpower, into the main channel at the optical switch position. In thepreferred embodiment, the AOM 219 is controlled to produce an opticalswitch pattern as described in FIG. 9 b, although any of the opticalswitch methods previously described could be implemented. The lens 213below the cartridge 210 images the 488 nm excitation illumination onto aphotodiode 223. The signal detected by this photodiode 223 is used tohelp distinguish fluorescently labeled cells from smaller debris thatmay carry the fluorescent label, and also to identify clumps of cellsthat might have formed. These events are rejected as candidates forsorting to the target output channel.

Yet another preferred embodiment would incorporate appropriate imagingand optical filtering to provide a forward scattering signal based onthe illumination of the cell by the 488 nm laser that is used to excitethe fluorescence. The optics would provide a range of angularsensitivity, such as, but not limited to this range, 0.8° to 10°, forthe detection of the forward scattering signal. This signal can helpcharacterize cells in addition to the fluorescence signal, as well ashelp distinguish cells from debris. The forward scattering illuminationis not limited to the fluorescence excitation laser, but could be at anyother wavelength provided by an additional light source that is properlyimaged into the main channel.

Yet another preferred embodiment would incorporate additionalfluorescence detection channels that are sensitive to fluorescenceemissions at different wavelength, typically using a single excitationwavelength, such as, but not limited to, 488 nm. Each detection channelwould incorporate a PMT with an appropriate dichroic mirror and emissionfilter for the fluorescence emission wavelength of the additionalfluorophore. From two to four fluorescence detection channels arereadily accommodated in this manner. Using more than one fluorophore inthis manner provides the ability for multiple detection criteria toidentify the target cells for sorting with the optical switch.

Yet another preferred embodiment would incorporate an error checkingcapability that provides optical illumination, typically as a narrowline across one of the channels in the network, and typically at alonger wavelength, perhaps, but not limited to, 785 nm from a solidstate laser, that is outside the range of wavelengths used forfluorescence detection and forward scatter detection, but is shorterthan the optical switch wavelength that is typically at 1070 nm. Thissource can be appropriately imaged into the microfluidic channel networkto provide lines that can be used to detect passage of particles throughany vertical plane in the network. This provides additional ability tocheck the performance of the optical switch performance and providesadditional capability for the timing of the trigger of the opticalswitch, as described in FIG. 11.

Yet another preferred embodiment of the optical system would incorporatean additional optical illumination path at, but not restricted to, 750nm, e.g., as produced by band pass filtering the light from an LED, andilluminating a region of the microfluidic channels with that light. Thatregion would be imaged through a 750 nm pass filter onto a CCD camera toprovide visualization of the performance of the cells flowing in themicrofluidic channel network at the bifurcation junction and/or at thepinch junction. The filters before the camera would be adequate to blockany shorter wavelength radiation associated with the excitation ordetection of fluorescence and with the forward/side scatter optics andthe error detection optics. The filters would also block the longerwavelength, 1070 nm light from the optical switch.

The preferred embodiment of the cartridge 210 shown in FIG. 20 isdesigned to hold the microfluidic channel network in a horizontalconfiguration, so that all of the channels and inlet/outlet ports are atthe same vertical level. This minimizes the effects of gravity on thepressure drops through the microfluidic channels, leading to more stableand controllable flow in the network. However, gravity will still havean effect on the cells in the flow, particularly as the cells pass fromthe cell sample reservoir into the cell inlet microfluidic channel.Another preferred embodiment of the sorter, to help control the effectsof gravity on settling of the cells in this reservoir and on theirsettling in the relatively slower flow in the inlet microfluidic channelbefore the cells flow speeds up at the pinch junction, is to enhance thebuoyancy of the cells, such that settling of the cells is minimized.Increasing the buoyancy can be achieved by using additives in the samplebuffer. Examples of these rheological control additives, particularlythose that are either pseudoplastic or shear thinning, or both, arexanthan gum, carageenan, sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose,hydroxypropylcellulose, hydroxypropyl guar, Gum Arabic, Gum Tragacanth,Alginate, polyacrylates, carbomer. Other additives include Histopaque™,which is a mixture of polysucrose and sodium diatrizoate, and Optiprep™,which is a 60% w/v solution of iodixanol in water. The concentration ofthese additives used depends on the density of the cell being sorted.For instance, in the case of Optiprep™ the concentration can range from5% to 40%. Finally, salinity of the sample buffer and addition ofsucrose can also be used to adjust the buoyancy of cells.

The buffers that are used for the cell sample volume and for the sheathflow can be any buffers that are biologically compatible with the cellsthat are being sorted, and are compatible with optical illumination thatis used both for the fluorescence detection modality and for the opticalswitch, i.e., the buffer has sufficiently low absorbance at thefluorescence excitation/detection wavelengths and the optical switchwavelength. A preferred embodiment of the sheath buffer uses PBS/BSA,phosphate buffered saline (PBS) at pH 7.2 with 1% bovine serum albumin(BSA) fraction 5. A preferred embodiment of the cell buffer uses PBS/BSAwith 14.5% Optiprep for live cell samples and 27% Optiprep for a varietyof formalin fixed cell samples.

The performance of the optical switch method of cell sorting in amicrofluidic channel network is evaluated by the throughput, purity andrecovery of the sort as previously described. The cartridge described inFIG. 20 is optimized to allow measurement of the performance, since itis made of acrylic, the bottoms of the target and waste collectionreservoirs are transparent and the cells that are sorted into thesereservoirs can be quantified as to both number and fluorescence labelingusing an inverted fluorescence microscope. Several of the switchingconfigurations described in FIGS. 3-11 were evaluated. These evaluationswere performed using a 50:50 mix of live HeLa:HeLa-GFP cells that wassorted using either a 1- or 2-sided stationary laser spot, or a 0° or 8°1-sided laser sweep. The laser was swept at 240 Hz. The laser ON timewas 4 msec and the laser power was 20 W for all switch modes. For theswept spot method, the focused IR laser spot was translated about 70 μmalong the main channel.

As shown in FIG. 22, the bi-directional optical switch with laser spots,described in FIG. 6, gave good results for purity and recovery for the50:50 mixes of target:non-target cells up to a throughput of 50 cells/s.However, at lower subpopulation concentrations (data not shown), it wasnot an efficient use of laser power to switch non-target cells, andcoincidence errors increased at higher cell throughput rates.Additionally, small particulates that were not switched wouldcontaminate the target reservoir.

FIG. 22 also shows the performance of the 1-sided switching methods,described in FIG. 9, with a stationary laser spot or a spot that istranslated in the direction of flow, either parallel or at a slightangle to the flow. The sample core flow stream was biased to the wasteoutlet such that all the cells went to waste by default in the absenceof the optical switch. Both of these methods gave improved performance,as shown by the plots. The fact that the performance of these twomethods crosses, suggests that the triggering of the optical switch wasnot optimal, and suggests that the active triggering of the opticalswitch as described in FIGS. 10 and 11 will improve performance.

ENDNOTES

-   1. H. M. Shapiro, Practical flow cytometry, Wiley-Liss, New York,    2003.-   2. Y. Fu, C. Spence, A. Scherer, F. H. Arnold and S. R. A. Quake,    “Microfabricated fluorescence-activated cell sorter,” Nat.    Biotechnol. 17, pp. 1109-1111, 1999.-   3. Y. Fu, H.-P. Chou, C. Spence, F. H. Arnold, and S. R. Quake, “An    integrated microfabricated cell sorter,” Anal. Chem. 74, pp.    2451-2457, 2002.-   4. Wolff, I. R. Perch-Nielsen, U. D. Larsen, P. Friis, G.    Goranovic, C. R. Poulsen, J. P. Kutter and P. Telleman, “Integrating    advanced functionality in a microfabricated high-throughput    fluorescent-activated cell sorter,” Lab Chip 3, pp. 22-27, 2003-   5. Li, P. C. H. & Harrison, D. J. Transport, manipulation, and    reaction of biological cells on-chip using electrokinetic effects.    Anal. Chem. 69, 1564-1568 (1997).-   6. Dittrich, P. S. & Schwille, P. An integrated microfluidic system    for reaction, high-sensitivity detection, and sorting of fluorescent    cells and particles. Anal. Chem. 75, 5767-5774 (2003).-   7. Fiedler, S., Shirley, S. G., Schnelle, T. & Fuhr, G.    Dielectrophoretic sorting of particles and cells in a microsystem.    Anal. Chem. 70, 1909-1915 (1998).-   8. Y. Huang, K. L. Ewalt, M. Tirado, R. Haigis, A. Forster, D.    Ackley, M. J. Heller, J. P. O'Connell, M. T. Krihak, “Electric    manipulation of bioparticles and macromolecules on microfabricated    electrodes,” Anal. Chem. 73, pp. 1549-1559, 2001.-   9. M. Durr, J. Kentsch, T. Muller, T. Schnelle and M. Stelzle,    “Microdevices for manipulation and accumulation of micro- and    nanoparticles by dielectrophoresis,” Electrophoresis 24, pp.    722-731, 2003.-   10. T. N. Buican, M. J. Smyth, H. A. Crissman, G. C. Salzman, C. C.    Stewart and J. C. Martin, “Automated single-cell manipulation and    sorting by light trapping,” Appl. Opt. 26, pp. 5311-5316, 1987.

1. A method for cell sorting in a device comprising a microfluidicnetwork having a sample inlet, one or more inlet channels, a mainchannel, and at least two outlet channels, comprising the steps of:receiving cells in a fluidic medium at the sample inlet, flowing thecells through the main channel, subjecting the cells to a bias flowpreferentially resulting in collection of the cells in a first reservoirin fluid communication with a first outlet channel, wherein the biasflow is created by applying asymmetric pressure to one or more of thechannels of the microfluidic network, identifying a cell to be sortedthrough application of a lateral force into a second reservoir coupledto a second outlet channel, and applying a lateral force on the cell,the lateral force configured to be translated at a non-zero angle downthe main channel toward the at least two outlet channels andcharacterized in that the lateral force is a non-trapping force, wherebythe cell is moved transverse to the preferentially biased flow andselectively exits the second outlet channel coupled to the secondreservoir.
 2. The method for cell sorting of claim 1, wherein subjectingthe cell to a bias flow by applying asymmetric pressure comprisesintroducing a pressure differential across the at least two outletchannels to direct the cell toward the first outlet channel coupled tothe first reservoir.
 3. The method for cell sorting of claim 2, furthercomprising separately controlling the pressure at the at least twooutlet channels to create the pressure differential, wherein thepressure at each of the at least two outlet channels is selected fromthe group consisting of: above atmospheric pressure, atmosphericpressure, and below atmospheric pressure.
 4. The method for cell sortingof claim 1, wherein subjecting the cell to a bias flow by applyingasymmetric pressure comprises introducing a pressure differential byusing at least one pneumatic controller to separately control pressureat one or more of the outlet channels, the sample inlet channel and theinlet channels.
 5. The method for cell sorting of claim 1, furthercomprising at least two inlet channels wherein subjecting the cell to abias flow by applying asymmetric pressure comprises introducing apressure differential across the at least two inlet channels to directthe cell toward the first outlet channel coupled to the first reservoir.6. The method for cell sorting of claim 5, further comprising separatelycontrolling the pressure at the at least two inlet channels to createthe pressure differential, wherein the pressure at each of the at leasttwo inlet channels is selected from the group consisting of: aboveatmospheric pressure, atmospheric pressure, and below atmosphericpressure.
 7. The method for cell sorting of claim 1, wherein the firstreservoir is a non-target cell reservoir.
 8. The method for cell sortingof claim 1, wherein the second reservoir is a target cell reservoir. 9.A method for cell sorting in a device comprising a microfluidic networkhaving a sample inlet, one or more inlet channels, a main channel, andat least two outlet channels, comprising the steps of: receiving cellsin a fluidic medium at the sample inlet, flowing the cells through themain channel, subjecting the cells to a bias flow preferentiallyresulting in collection of the cells in a first reservoir in fluidcommunication with a first outlet channel, wherein the bias flow iscreated by configuring one or more of the channels of the microfluidicnetwork to have asymmetric geometries, identifying a cell to be sortedthrough application of a lateral force into a second reservoir coupledto a second outlet channel, and applying a lateral force on the cell,configured to be translated at a non-zero angle down the main channeltoward the at least two outlet channels and characterized in that thelateral force is a non-trapping force, whereby the cell is movedtransverse to the preferentially biased flow and selectively exits thesecond outlet channel coupled to the second reservoir.
 10. The method ofcell sorting of claim 9, wherein the bias flow is created by configuringthe at least two outlet channels to have asymmetric geometries.
 11. Themethod of cell sorting of claim 10, wherein creating the bias flow byconfiguring the at least two outlet channels to have asymmetricgeometries comprises configuring the first outlet channel to have alarger volumetric flow relative to the second outlet channel.
 12. Themethod of cell sorting of claim 11, wherein configuring the first outletchannel to have a larger volumetric flow relative to the second outletchannel comprises configuring the first outlet channel to have a largerchannel width relative to the second outlet channel.
 13. The method ofcell sorting of claim 9, further comprising at least two fluid inletchannels wherein the bias flow is created by configuring the first andsecond inlet channels to have asymmetric geometries.
 14. The method ofcell sorting of claim 13, wherein creating the bias flow by configuringthe first and second inlet channels to have asymmetric geometriescomprises configuring a first fluid inlet channel to have a largervolumetric flow relative to a second fluid inlet channel.
 15. The methodof cell sorting of claim 14, wherein configuring the first inlet channelto have a larger volumetric flow relative to the second inlet channelcomprises configuring the first inlet channel to have a larger channelwidth relative to the second inlet channel.
 16. The method of cellsorting of claim 9, wherein the microfluidic network further comprisesat least two fluid inlet channels and wherein subjecting the cell to abias flow by configuring one or more of the channels of the microfluidicnetwork to have asymmetric geometries comprises configuring inletchannels to provide equal flow rates and configuring the first outletchannel to have a larger volumetric flow relative to the second outletchannel.
 17. The method of cell sorting of claim 9, wherein themicrofluidic network further comprises at least two fluid inlet channelsand wherein subjecting the cell to a bias flow by configuring one ormore of the channels of the microfluidic network to have asymmetricgeometries comprises configuring the inlet channels to have symmetricgeometries and configuring the outlet channels to have asymmetricgeometries.
 18. The method of cell sorting of claim 9, wherein the firstreservoir is a non-target cell reservoir.
 19. The method of cell sortingof claim 9, wherein the second reservoir is a target cell reservoir. 20.The method for cell sorting of claim 9, wherein identifying a cell to besorted comprises using at least one detection modality selected from thegroup comprising fluorescent and non-fluorescent detection modalities